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Azo Dye-Induced Alterations in Vitamin B-6 Metabolism and in Pyridoxal 5'-Phosphate-Binding Proteins in Rat Liver^1,2,

Department of Biochemistry, University of Vermont, College of Medicine, Burlington, VT 05405

The effects of the hepatocarcinogen, 3'-methyl-4-dimethylamino-azobenzene, on vitamin B-6 metabolism in rat liver were studied. The following parameters were measured: pyridoxal 5'-phosphate (PLP) concentrations in plasma, brain, liver and azo dye-induced hepatomas, as well as the activities of pyridoxine (PN) kinase, pyridoxine 5'-phosphate (PNP) oxidase, PNP phosphatase and PLP-dependent ornithine decarboxylase. Hepatomas more closely resembled fetal than normal adult rat liver with respect to their ability to convert vitamer forms such as PN to coenzymatically active PLP. Microtiter plate enzyme-linked immunosorbent analyses revealed that the absence of PNP oxidase activity in a dissectable hepatoma was attributable to the absence of enzyme protein. In addition, monoclonal antibodies to vitamin B-6 were used in a Western immunoblot technique to examine the effects of azo dye ingestion on the pattern of PLP-binding proteins in cytosolic extracts of liver and hepatomas. Nitrocellulose blots of electrophoretically resolved cytosolic extracts probed for PLP-binding proteins showed increasing complexity with development; hepatomas bore a striking resemblance to fetal liver. The data indicate that hepatomas lose the properties of terminally differentiated hepatic tissue and take on the properties of fetal hepatic tissue characterized by lower concentrations of PLP, selective use of the coenzyme, and a lowered-to-absent capability to convert precursor vitamer forms to PLP. Therefore, with respect to vitamin B-6 metabolism and use, it appears likely that azo dye-induced hepatocarcinogenesis involves proliferation of a stem cell type(s) having the phenotypic characteristics of fetal hepatic tissue.

KEY WORDS: • vitamin B-6 • hepatomas • pyridoxal 5'-phosphate-binding proteins

¹ This work was supported by U.S. Public Health Service Grant No. 35878 awarded by the National Cancer Institute, Department of Health and Human Services. A grant from the Pelham Parkway League for Grants in Cancer Research, Inc., enabled us to purchase a Hoefer PS 1200 DC power supply.

² A preliminary report of these data was presented at the Vitamin B6 Workshop, 1984, Banff Conference Centre, Banff, Alberta, Canada.

³ Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.

⁴ To whom correspondence should be addressed.

Manuscript received 19 August 1985. Revision accepted 20 November 1985.